RESUMO
Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles released by almost all cell types and can be found in biological fluids, such as blood and urine. EVs play an important role in various physiological and pathological processes via cell-cell communication, highlighting their potential applications as diagnostic markers for diseases and therapeutic drug delivery carriers. Although various methods have been developed for the isolation of EVs from biological fluids, most of them exhibit major limitations, including low purity, long processing times, and high cost. In this study, we developed a size-exclusion chromatography (SEC) column device using hydrophilic porous silica gel (PSG). Owing to the resistance to pressure of the device, a rapid system for EV isolation was developed by connecting it to a flash liquid chromatography system furnished with a UV detector and a fraction collector. This system can be used for the real-time monitoring of eluted EVs by UV absorption without further analysis and separation of high-purity EVs from urine samples with high durability, reusability, and reproducibility. In addition, there were no significant differences between the PSG column- and conventional SEC column-isolated EVs in the proteome profiles and cellular uptake activities, suggesting the good quality of the EVs isolated by the PSG column. These findings suggest that the PSG column device offers an effective and rapid method for the isolation of intact EVs from biological fluids.
Assuntos
Vesículas Extracelulares , Proteoma , Cromatografia em Gel , Vesículas Extracelulares/química , Bicamadas Lipídicas/metabolismo , Porosidade , Proteoma/análise , Reprodutibilidade dos Testes , Sílica GelRESUMO
We present an ultrasensitive immunoassay system for disease biomarkers utilizing the immuno-wall device and an enzymatic amplification reaction. The immuno-wall device consisted of 40 microchannels, each of which contained an antibody-modified wall-like structure along the longitudinal axis of the microchannel. The wall was fabricated with a water-soluble photopolymer containing streptavidin by photolithography, and biotinylated capture antibodies were immobilized on the sides through streptavidin-biotin interaction. For an assay, introducing the target biomarker and secondary and labeled antibodies produced a sandwich complex anchored on the sides of the wall. A conventional immuno-wall device uses a fluorescence-labeled antibody as a labeling antibody. To achieve an ultrasensitive detection of a trace biomarker, we used an enzyme label and amplified the signal with the enzymatic reaction with a fluorogenic substrate in the microchannel. The highest signal/background ratio was obtained by using alkaline phosphatase-labeled antibody and 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate. To evaluate the device performance, we detected human C-reactive protein (CRP) as a model biomarker. The detection limit (LOD) of CRP in phosphate-buffered saline was 2.5 pg mL-1 with a sample volume of 0.25 µL. This LOD was approximately 3 orders of magnitude lower than that obtained with fluorescent-dye (DyLight 650)-labeled antibody. In addition, the present device provided a wide detection range of 0.0025-10 ng mL-1 for CRP. We successfully developed an ultrasensitive immunoassay system with simple operation and only a small sample volume.
Assuntos
Proteína C-Reativa/análise , Imunoensaio/métodos , Dispositivos Lab-On-A-Chip , Acridinas/química , Fosfatase Alcalina/química , Animais , Anticorpos Imobilizados/imunologia , Biomarcadores/sangue , Proteína C-Reativa/imunologia , Corantes Fluorescentes/química , Cabras , Humanos , Imunoensaio/instrumentação , Limite de Detecção , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , CoelhosRESUMO
Various liquid biopsy methods have been developed for the non-invasive and early detection of diseases. In particular, the detection of circulating tumor cells (CTCs) and cancer-associated fibroblasts (CAFs) in blood has been receiving a great deal of attention. We have been developing systems and materials to facilitate such liquid biopsies. In this study, we further developed glass filters (with various patterns of holes, pitches, and non-adhesive coating) that can capture CTCs, but not white blood cells. We optimized the glass filters to capture CTCs, and demonstrated that they could be used to detect CTCs from lung cancer patients. We also used the optimized glass filters for detecting CAFs. Additionally, we further developed a system for visualizing the captured cells on the glass filters. Finally, we demonstrated that we could directly culture the captured cells on the glass filters. Based on these results, our high-performance glass filters appear to be useful for capturing and culturing CTCs and CAFs for further examinations.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Humanos , Células Neoplásicas Circulantes/patologia , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologiaRESUMO
Considering the challenges in isolating circulating tumor cells (CTCs) pertaining to cellular stress and purity, we report the application of a blood microfiltration device as an optimal approach for noninvasive liquid biopsy to target CTCs. We experimentally analyzed the filtration behavior of the microfilter using pressure sensing to separate tumor cells from leukocytes in whole blood. This approach achieved an average recovery of >96% of the spiked tumor cells and depletion of >99% of total leukocytes. Furthermore, we carried out genomic profiling of the CTCs using the blood microfiltration device. The method was also applied in a clinical setting; DNA amplification was performed using a small number of microfiltered CTCs and epidermal growth factor receptor mutations were successfully detected to characterize the efficacy of molecularly targeted drugs against lung cancer. Overall, the proposed method can provide a tool for evaluating efficient filtration pressure to concentrate CTCs from whole blood.
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Cancer-derived small extracellular vesicles (sEVs) are multifunctional particles with a lipid bilayer structure that are involved in cancer progression, such as malignant proliferation, distant metastasis, and cancer immunity evasion. The separation protocol used to isolate sEVs is an important process and thus, several have been developed, including ultracentrifugation (UC), size exclusion chromatography (SEC), and affinity purification using antibodies against sEV surface antigens. However, the effects of different separation methods on sEV components have not been adequately examined. Here, we developed a semi-automated system for collecting sEVs by combining SEC and preparative high-performance liquid chromatography and applied it to metabolome analysis. The developed SEC system could recover sEVs more efficiently and non-destructively than UC, suggesting that it is an appropriate recovery method for metabolic analysis and reflects biological conditions. Furthermore, using the developed SEC system, we performed metabolome analysis of sEVs from isocitrate dehydrogenase 1 (IDH)-mutated human colon HCT116 cells, which produce the oncogenic metabolite, 2-hydroxyglutaric acid (2-HG). IDH1-mutated HCT116 cells released significantly more sEVs than wild-type (WT) cells. The metabolomic profiles of IDH1 mutant and WT cells showed distinct differences between the cells and their sEVs. Notably, in IDH mutant cells, large amounts of 2-HG were detected not only in cells, but also in sEVs. These results indicate that the SEC system we developed has wide potential applications in sEVs research.
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A solid-state molecular beacon using a gold support as a fluorescence quencher is combined with a polydimethylsiloxane (PDMS) microfluidic channel to construct an optical sensor for detecting single-stranded DNA binding protein (SSBP) and histone protein. The single-stranded DNA-Cy3 probe or double-stranded DNA-Cy3 probe immobilized on the gold surface is prepared for the detection of SSBP or histone, respectively. Due to the different quenching ability of gold to the immobilized single-stranded DNA-Cy3 probe and the immobilized double-stranded DNA-Cy3 probe, low fluorescence intensity of the attached single-stranded DNA-Cy3 is obtained in SSBP detection, whereas high fluorescence intensity of the attached double-stranded DNA-Cy3 is obtained in histone detection. The amounts of SSBP in sample solutions are determined from the degree of fluorescence recovery of the immobilized single-stranded DNA-Cy3 probe, whereas that of histone in sample solutions is determined from the degree of fluorescence quenching of the immobilized double-stranded DNA-Cy3 probe. Using this approach, label-free detection of target proteins at nanomolar concentrations is achieved in a convenient, general, continuous flow format. Our approach has high potential for the highly sensitive label-free detection of various proteins based on binding-induced conformation changes of immobilized DNA probes.
Assuntos
Técnicas Biossensoriais/instrumentação , Sondas de DNA/química , Sondas de DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Sondas de Oligonucleotídeos/química , Sondas de Oligonucleotídeos/metabolismo , Sequência de Bases , Sondas de DNA/genética , Histonas/metabolismo , Sondas de Oligonucleotídeos/genética , Espectrometria de Fluorescência , Fatores de TempoRESUMO
The study of the multimolecular crowding around cancer cells has opened up the possibility of developing new devices for cancer diagnosis and analysis through the measurement of intercellular communication related to cell proliferation and invasive metastasis associated with cancer malignancy. In particular, cells and extracellular vesicles that flow into the bloodstream contain metabolites and secreted products of the cancer microenvironment. These are positioned as targets for the development of new devices for the understanding and application of multimolecular crowding around cancer cells. Examples include the separation analysis of cancer cells in blood for the next generation of less invasive testing techniques, and mapping analysis using Raman scattering to detect cancer cells without staining. Another example is the evaluation of the relationship between exosomes and cancer traits for the exploration of new anti-cancer drugs, and the commercialization of exosome separation devices for ultra-early cancer diagnosis. The development of nanobiodevice engineering, which applies multimolecular crowding to conventional nanobioscience, is expected to contribute to the diagnosis and analysis of various diseases in the future.
Assuntos
Separação Celular/métodos , Neoplasias/diagnóstico , Separação Celular/instrumentação , Exossomos , Humanos , Neoplasias/sangue , Células Neoplásicas Circulantes , Análise Espectral RamanRESUMO
Recent studies have shown that extracellular vesicles (EVs) can be utilized as appropriate and highly specific biomarkers in liquid biopsy for the diagnosis and prognosis of serious illness. However, there are few methods that can collect and isolate miRNA in EVs simply, quickly and efficiently using general equipment such as a normal centrifuge. In this paper, we developed an advanced glass membrane column (AGC) device incorporating a size-controlled macro-porous glass (MPG) membrane with a co-continuous structure to overcome the limitations of conventional EV collection and miRNA extraction from the EVs. The size of macro-pores in the MPG membrane could be accurately controlled by changing the heating temperature and time on the basis of spinodal decomposition of B2O3, Na2O, and SiO2 in phase separation. The AGC device with an MPG membrane could collect the EVs simply and quickly (< 10 min) from cell culture supernatant, serum and urine. This AGC device could extract miRNA from the EVs captured in the MPG membrane with high efficiency when combined with a miRNA extraction solution. We suggest that the AGC device with an MPG membrane can be useful for the diagnosis and prognosis of serious illness using of EVs in various kinds of body fluids.
Assuntos
Vesículas Extracelulares/genética , MicroRNAs/isolamento & purificação , Vesículas Extracelulares/química , Vesículas Extracelulares/ultraestrutura , Vidro , Células Hep G2 , Humanos , Biópsia Líquida/métodos , Membranas , MicroRNAs/urina , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , PorosidadeRESUMO
We have developed a fluorescence resonance energy transfer (FRET) probe based on the conjugation of a quantum dot (QD) with dye (YOYO-3) intercalated DNA. The FRET-inducing electrostatic coupling of DNA and the QD made structural changes to the QD-DNA conjugates, which significantly prevented an enzymatic reaction between the DNA and a conventional restriction endonuclease (EcoRI).
Assuntos
DNA/química , Endonucleases/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Oxazóis/química , Pontos Quânticos , Substâncias Intercalantes/química , Sensibilidade e Especificidade , Eletricidade EstáticaRESUMO
Positioning single cells on a solid surface is a crucial technique for understanding the cellular functions and cell-cell interactions in cell culture assays. We developed a microfluidic chip for depositing single cells in microwells using a simple micropipette operation. Cells were delivered to microwells by the meniscus motion of liquid interface. The residue deposits of cells were redistributed with air injection, and the isolated single cells were stored in microwells. Different microwell sizes and depths were studied to evaluate the trapping possibility of cells. Medium replacement and cell viability staining with the isolated single cells were achieved in microwells. The chip will serve as a tool for single-cell patterning in an easy-to-use manner.
RESUMO
The method described here enables the automatic stretching and patterning of single DNA molecules onto a solid surface. It does not require chemical modification of the DNA or surface modification of the substrate. To detect a signal variation caused by sequence-specific dye binding or partial melting, it is crucial that the DNA molecules are arrayed in a parallel direction inside the narrow microscopic field. The method uses zigzag-shaped microgrooves in a densely-arranged molecular patterning apparatus in a microfluidic channel. By syringing through the microchannel, over 1500 DNA molecules can be arrayed simultaneously in the microgrooves. It will therefore serve as a template preparation for DNA molecular diagnosis by high-resolution imaging.
Assuntos
DNA , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Técnicas de Diagnóstico Molecular/métodos , Imagem Individual de Molécula/métodos , Desenho de Equipamento , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Diagnóstico Molecular/instrumentação , Nanotecnologia , Imagem Individual de Molécula/instrumentaçãoRESUMO
We investigated the transduction function of a cationic dextran hydroxypropyltrimethyl ammonium chloride-coated magnetic iron oxide nanoparticle (TMADM-03) for transducing quantum dots (QDs) into adipose tissue-derived stem cells (ASCs). As a result, the fluorescence intensity of ASCs labeled with QDs using TMADM-03 was much higher than that of QDs only labeling. These data suggest that TMADM-03 can be useful as a transduction agent for QDs in stem-cell imaging.
Assuntos
Dextranos/química , Compostos Férricos/química , Nanopartículas Metálicas/química , Imagem Molecular/métodos , Imagem Óptica/métodos , Propanóis/química , Compostos de Amônio Quaternário/química , Células-Tronco/citologia , Tecido Adiposo/citologia , Animais , Feminino , Camundongos , Pontos Quânticos/químicaRESUMO
The steady state and time-resolved photoluminescence quenching of streptavidin modified CdSe/ZnS quantum dots (QDs) instigated by biotin-peptide-BHQ-1 (biotin-pep-BHQ-1) molecule was investigated. Here, we have achieved an efficient photoluminescence (PL) quenching of QDs with the conjugation of dark quencher (black hole quencher-BHQ) molecules intermediated with the GPLGVRGK peptide. The luminescence of streptavidin-QDs585 was decreased upon titration with a nano molar concentration of the biotin-GPLGVRGK-BHQ-1 molecule. It has been suggested that the decrease of QDs PL occurred through a Förster resonance energy transfer (FRET) mechanism from the analysis of steady state photoluminescence intensity measurements as well as time resolved lifetime measurements of streptavidin-QDs and QDs-(pep-BHQ-1)n conjugates. The sequence of intermediate peptide GPLG↓VRGK can act as a target material for matrix metalloproteinases-2 (MMP-2) produced by cancer cells at its Gly and Val region, shown by the down-headed arrow. Interestingly, here the reported self-assembled QDs-(pep-BHQ-1)n conjugates could detect the presence MMP-2 at a detection limit of 1 ng/mL with a clear luminescence recovery.
Assuntos
Compostos de Cádmio/química , Transferência Ressonante de Energia de Fluorescência , Metaloproteinase 2 da Matriz/análise , Peptídeos/química , Pontos Quânticos/química , Compostos de Selênio/química , Sulfetos/química , Compostos de Zinco/química , Medições Luminescentes , Metaloproteinase 2 da Matriz/química , Modelos Moleculares , Conformação Proteica , Estreptavidina/químicaRESUMO
Abnormal DNA methylation in CpG-rich promoters is recognized as a distinct molecular feature of precursor lesions to cancer. Such unintended methylation can occur during in vitro differentiation of stem cells. It takes place in a subset of genes during the differentiation or expansion of stem cell derivatives under general culture conditions, which may need to be monitored in future cell transplantation studies. Here we demonstrate a microfluidic device for investigating morphological length changes in DNA methylation. Arrayed polymer chains of single DNA molecules were fluorescently observed by parallel trapping and stretching in the microfluidic channel. This observational study revealed that the shortened DNA length is due to the increased rigidity of the methylated DNA molecule. The trapping rate of the device for DNA molecules was substantially unaffected by changes in the CpG methylation.
RESUMO
The facile synthesis of ZnS-AgInS2 (ZAIS) as cadmium-free QDs and their application, mainly in solar cells, has been reported by our groups. In the present study, we investigated the safety and the usefulness for labeling and in vivo imaging of a newly synthesized aqueous ZnS-coated ZAIS (ZnS-ZAIS) carboxylated nanoparticles (ZZC) to stem cells. ZZC shows the strong fluorescence in aqueous solutions such as PBS and cell culture medium, and a complex of ZZC and octa-arginine (R8) peptides (R8-ZZC) can achieve the highly efficient labeling of adipose tissue-derived stem cells (ASCs). The cytotoxicity of R8-ZZC to ASCs was found to be extremely low in comparison to that of CdSe-based QDs, and R8-ZZC was confirmed to have no influence on the proliferation rate or the differentiation ability of ASCs. Moreover, R8-ZZC was not found to induce the production of major inflammatory cytokines (TNF-α, IFN-γ, IL-12p70, IL-6 and MCP-1) in ASCs. Transplanted R8-ZZC-labeled ASCs could be quantitatively detected in the lungs and liver mainly using an in vivo imaging system. In addition, high-speed multiphoton confocal laser microscopy revealed the presence of aggregates of transplanted ASCs at many sites in the lungs, whereas individual ASCs were found to have accumulated in the liver.
Assuntos
Tecido Adiposo/citologia , Imagem Óptica/métodos , Pontos Quânticos , Coloração e Rotulagem/métodos , Transplante de Células-Tronco , Células-Tronco/fisiologia , Animais , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/toxicidade , Camundongos Endogâmicos C57BL , Imagem Corporal TotalRESUMO
Nano- and microhydrogels were fabricated in water by first to third generation triethoxysilyl focal poly(amido amine) dendrons with hexyl spacer. The focal points of dendrimers were hydrolyzed and covalent-bonded through the simple sol-gel process at an acidic or basic catalytic condition. The growth of aggregates and the following gel formation were determined by rapid increase and convergent steady value in light scattering of dendron solutions. The sol-gel reaction was also confirmed from the disappearance of an infrared absorption band of Si-O(CH) stretching vibration mode (1080 cm) and the appearance of Si-O-Si stretching bands (1136 and 1049 cm). The resultant gels were transparent and rather fluid. Transmission electron microscopic images of the gels showed three-dimensional dendritic growing of fine fibrils. The nanogel nuclei grew up favourably to nanogels in acidic conditions and to microgels in basic conditions, and the growth was more remarkable at higher generation of dendrimers. At high concentration of dendrimer, macrogels with fiber-like texture were formed. It was supported that siloxane-linked focal groups constructed main chains and branches of fibrils, and dendron side chains coated polysiloxane backbones. The hydrogels emitted fluorescence, which was stronger at base-catalyzed condition than at acid-catalyzed condition. This indicates that crowded circumstances or large amount of fluorescence-inducing moieties intensify the fluorescence. Fluorescent images of such architectures were visualized on a fluorescent microscope.
RESUMO
A field of recent diagnostics and therapeutics has been advanced with quantum dots (QDs). QDs have developed into new formats of biomolecular sensing to push the limits of detection in biology and medicine. QDs can be also utilized as bio-probes or labels for biological imaging of living cells and tissues. More recently, QDs has been demonstrated to construct a multifunctional nanoplatform, where the QDs serve not only as an imaging agent, but also a nanoscaffold for diagnostic and therapeutic modalities. This review highlights the promising applications of multi-functionalized QDs as advanced nanosensors for diagnosing cancer and as innovative fluorescence probes for in vitro or in vivo stem cell imaging in regenerative medicine.
Assuntos
Neoplasias/diagnóstico , Pontos Quânticos , Transplante de Células-Tronco , Animais , Diagnóstico por Imagem , Corantes Fluorescentes/uso terapêutico , Humanos , Pontos Quânticos/uso terapêutico , Medicina Regenerativa/métodosRESUMO
Quantum dots (QDs) have recently been investigated as fluorescent probes for detecting a very small number of biomolecules and live cells; however, the establishment of molecular imaging technology with on-off control of QD fluorescence remains to be established. Here we have achieved the fluorescence off state of QDs with the conjugation of black hole quencher (BHQ) molecules intermediated with peptide by using streptavidin-QDs585 and biotin-pep-BHQ-1. The fluorescence of streptavidin-QDs585 was decreased by the addition of biotin-pep-BHQ-1 in a dose-dependent manner. It has been suggested that the decrease in QDs585 fluorescence occurred through a Förster resonance energy transfer (FRET) mechanism from the analysis of fluorescence intensity and lifetime of streptavidin-QDs585 and QDs585-pep-BHQ-1. QDs585 fluorescence could be quenched by more than 60% efficiency in this system. The sequence of intermediate peptide (pep) was GPLGVRGK, which can be cleaved by matrix metalloproteinases (MMPs) produced by cancer cells. QDs585-pep-BHQ-1 is thus expected to detect the MMP production by the recovery of QDs585 fluorescence as a new bioanalytical agent for molecular imaging.
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The molecular stretching of DNA is an indispensable tool for the optical exploration of base sequences and epigenomic changes of DNA at a single molecule level. In stretching terminal-unmodified DNA molecules parallel to each other on solid substrate, the receding meniscus assembly and capillary force through the dewetting process are quite useful. These can be achieved by pulling the substrate out of the DNA solution or sliding a droplet of DNA solution between a pair of substrates. However, currently used methods do not allow control over liquid interface motion and single-molecule DNA positioning. Here, we show a microfluidic device for stretching DNA molecules by syringing through microgrooves. The device can trap single DNA molecules at vertices of the microgrooves, which were designed as parallel zigzag lines. Different zigzag pattern depths, sizes, and shapes were studied to evaluate the adsorption possibility of DNA on the surface. The microfluidic transfer of the liquid interface stretched over 1500 DNA molecules simultaneously. The stretched DNA molecules could be stamped to a silanized surface. The device will therefore serve as a template preparation for high-resolution DNA imaging studies.
Assuntos
DNA/química , Técnicas Analíticas Microfluídicas/métodos , Corantes Fluorescentes , Técnicas Analíticas Microfluídicas/instrumentação , Conformação de Ácido NucleicoRESUMO
We reported an optical DNA/protein microfluidic sensor which consists of single stranded (ss) DNA-Cy3 probes on gold surface and simple line-shape microfluidic channel. These ssDNA-Cy3 probes with random sequence in bulk solution or on gold surface exhibits fluorescence enhancement after binding with complementary ssDNA (cssDNA) targets. Particularly it did not require complicated design or hairpin-like stem-loop conformation, which made it easier to be made and applied in analytes detection by fluorescence switching techniques. Using ssDNA-cy3 probes attached on gold surface in a microfluidic channel, strong fluorescence enhancement was measured by ssDNA with cssDNA binding or ssDNA with cssDNA-biotin binding. The following introduction of streptavidin resulted in fluorescence quenching (fluorescence decrease) because of the binding of hybridized DNA-biotin with streptavidin. This sensor showed strong affinity and high sensitivity toward the streptavidin, the minimum detectable concentration for streptavidin was 1 pM, equating to an absolute detection limit of 60 amol in this microfluidic channel. Microfluidic channel height and flow rate is optimized to increase surface reaction efficiency and fluorescence switching efficiency. In contrast to previously reported optical molecular beacon approach, this sensor can be used not only for the detection of cssDNA target, but also for the detection of streptavidin. This microfluidic sensor offers the promise of analyzing kinds of molecular targets or immunoreactions.